The specific aim of this project is to provide the time and opportunity for the PI to learn how to utilize acute slice and organotypic slice culture preparations within the context of physiological regulation of neuropeptide gene expression. The long term goal of the associated research project is to identify the afferent signalling mechanisms, cellular responses, and topography of the limbic forebrain neural circuits that help regulate homeostasis in the rat. These circuits are critical for regulating the behavioral, autonomic and endocrine response of the animal to homeostatic disturbance. The central hypothesis is that homeostatic disturbances modify chemically-coded information contained within neurons by modulating the mRNAs that code for singly- and co- expressed neuropeptides. These cell- and stimulus-specific modifications facilitate the appropriate response by tile animal, either by modulating the activity of the autonomic nervous system, neuroendocrine function, or perhaps by modifying the central pattern generators that develop and regulate goal-directed behaviors. Because of the relative simplicity of the underlying physiology and behavior, along with an extensive literature, the overall project concentrates on investigating the organization of the circuits and mechanisms controlling fluid balance in the rat. Within this context, the goals of this project are to learn how to use in vitro techniques to investigate the underlying cellular mechanisms which modulate peptide gene expression in the hypothalamic paraventricular nucleus (PVH) and central nucleus of the amygdala (CEA) after alterations in fluid homeostasis in the rat. Relating these results to important and well documented models allows the interpretation of data within a compelling and contextual framework not possible with many other currently used 'stress' models. The assay methods used-principally in situ hybridization and immunocytochemistry-will allow the detection of changes in CRH, proenkephalin and neurotensin mRNAs in the PVH and CEA after glucocorticoid or transmitter manipulation. With these in vitro techniques, the PI will learn to use conventional pharmacology and recently introduced methods of antisense oligonucleotide blockade of receptor translation to this end. A further goal is to see how other in vitro techniques (eg. electrophysiology, molecular techniques) might be adapted to the PI's research project. Collaborations, consultations, and interactions with expert faculty in the PI's institution, and elsewhere in Southern California will provide the expertise to complete the program. Facilities will be provided by the PI's institution. In the long term, investigating the topography and mechanisms that organize the homeostatic aspects of fluid balance will provide a working model for understanding the neural organization of a relatively simple mammalian behavior at the cellular, systems and behavioral level. It will eventually allow us to address many of the clinical disorders (eg. hypertension, obesity, eating disorders) currently of central importance to human health, that have perturbed homeostatic regulation at the core of their etiology.